The complete nucleotide sequence of the chloroplast genome of the hardwood species Eucalyptus globulus is presented and compared with chloroplast genomes of tree and non-tree angiosperms and two softwood tree species. The 160 286 bp genome is similar in gene order to that of Nicotiana, with an inverted repeat (IR) (26 393 bp) separated by a large single copy (LSC) region of 89 012 bp and a small single copy region of 18 488 bp. There are 128 genes (112 individual gene species and 16 genes duplicated in the inverted repeat) coding for 30 transfer RNAs, 4 ribosomal RNAs and 78 proteins. One pseudogene (ψ-infA) and one pseudo-ycf (ψ-ycf15) were identified. The chloroplast genome of E. globulus is essentially co-linear with that of another hardwood tree species, Populus trichocarpa, except that the latter lacks rps16 and rpl32, and the IR has expanded in Populus to include rps19 (part of the LSC in E. globulus). Since the chloroplast genome of E. globulus is not significantly different from other tree and non-tree angiosperm taxa, a comparison of hardwood and softwood chloroplasts becomes, in essence, a comparison of angiosperm and gymnosperm chloroplasts. When compared with E. globulus, Pinus chloroplasts have a very small IR, two extra tRNAs and four additional photosynthetic genes, lack any functional ndh genes and have a significantly different genome arrangement. There does not appear to be any correlation between plant habit and chloroplast genome composition and arrangement.